Abhishek Mishra

Congenital heart defects (CHD) are the most common type of birth defects. Several human case studies and genetically altered animal models have identified abnormalities in the development of ventricular conduction system (VCS) in the heart. While cell-based therapies hold promise for treating CHDs, translational efforts are limited by the lack of suitable in vitro models for feasibility and safety studies. A better understanding of cell differentiation pathways can lead to development of cell-based therapies for individuals living with CHD/VCS disorders. Here, we describe a new and reproducible 3-D cell culture method for studying cardiac cell lineage differentiation in vitro. We used primary ventricular cells isolated from embryonic day 11.5 (E11.5) mouse embryos, which can differentiate into multiple cardiac cell types including VCS cells. We compared 3-D cultures with three types of basement membrane extracts (BME) for their abilities to support E11.5 ventricular cell differentiation. In addition, the effects of atrial natriuretic peptide (ANP) and an inhibitor for its high affinity receptor were tested on cell differentiation in 3-D cultures. Following the cell culture, protocols for immunofluorescence imaging, cell extraction and protein isolation from the 3-D culture matrix and in-cell western methods are described. Further, these approaches can be used to study the effects of various ligands and genetic interventions on VCS cell development. We propose that these methodologies may also be extended for differentiation studies using other sources of stem cells such as induced pluripotent stem cells.

After ejaculation, sperm show a limited capacity for transcription and translation. In the oviduct, most of the signalling in sperm is nongenomic and is mediated through membrane receptors. Studies have shown that the cation channel of sperm (CatSper), cAMP, cGMP, protein kinases, and tyrosine phosphorylation are involved in the nongenomic signalling of progesterone (P4) in sperm. However, it is not known whether there is an interplay between P4 and cannabinoid receptors 1 and 2 (CB1 and CB2), transient receptor potential vanilloid 1 (TRPV1), CatSper channels, cAMP, inositol trisphosphate receptor (IP3R), and mitogen‐activated protein kinase (MAPK); these potential regulators are involved in the regulation of capacitation and the acrosome reaction. In the present study, selective blockers of CB1, CB2, TRPV1, CatSper channels, cAMP, protein kinase A (PKA), IP3R, and MAPK were used to identify their involvement in P4‐mediated bull sperm capacitation and the acrosome reaction. Selective blocking of any one of the molecules caused a significant reduction in P4 signalling (p < 0.05). Interestingly, blocking these molecules in combination followed by treatment with P4 resulted in the complete absence of capacitation and the acrosome reaction. Blocking a single receptor was not able to eliminate the P4‐induced capacitation and the acrosome reaction. In addition to the CB1 and CB2 receptors, there may be other signalling pathways that mediate P4 signalling. In conclusion, P4 signalling exhibited interplay with the cannabinoid receptors. The regulation of sperm capacitation and the acrosome reaction also involved cAMP, PKA, l‐type and T‐type calcium channels, TRPV1, inositol trisphosphate, and MAPK.